Screening for targeted disruptors with an AlphaLISA protein-protein interaction assay

Abstract: For nearly two decades the scientific community has warned us of the need to be prepared for a pandemic, whose likelihood has increased due to several factors. A major part of this preparedness is our ability to quickly identify the cause of the emerging disease and its route of infection. Once these characteristics are identified our ability to quickly exploit this knowledge becomes paramount. To this end, drug repurposing provides the tools of approved or at least highly tested drugs that might be applicable to the treatment of the disease. Taking the example of SARS-CoV-2, it was quickly realized, that, similar to related corona viruses, the point of interaction with cells was angiotensin-converting enzyme 2 (ACE2). To search for disruptors of the protein-protein interaction (PPI) between ACE2 and the SAR-CoV-2 Spike Protein (S1) Receptor Binding Domain (RBD) an AlphaLISA assay was selected. This proximity-based assay has several advantages, including the commercial availability of streptavidin donor beads and protein A acceptor beads. These allow for testing of various candidate protein combinations with appropriate Avi and Fc tags. Using this approach, it should be possible to identify existing drugs that disrupt the interaction between the 2 proteins leading to a decrease in emission signal produced. For this AlphaLISA assay there exists a useful counter-screen that can be used to eliminate some false-positives that interfere with the AlphaLISA assay by, for example, quenching singlet oxygen or absorbing either excitation or emission light. We show the results of assay optimization by cross-titration of proteins involved in the interaction as well as proof of concept data showing disruption of PPI and the utility of the counter-screen. This assay provides the framework for developing any screen in which disruption of PPI is the desired outcome.