Hicham Zegzouti

Promega Corporation, R&D department, Madison, WI, USA.

Traditional immunoassays to study proteins involved in signaling, protein-protein interactions and protein degradation can be tedious, require multiple washing steps, and are not easily adaptable to high throughput screening (HTS) formats. Here, we describe a novel immunoassay approach based on NanoLuc luciferase fragment complementation technology. This bioluminescent immunoassay (Lumit) takes less than two hours to complete in a homogeneous “Add and Read” format and was successfully used to monitor the activation of multiple signaling pathways through specific nodes of phosphorylation (e.g., pERK, pAKT, and pSTAT3) in unmodified cells. We also tested deactivation of these pathways or degradation of specific endogenous targets with different small and large molecule inhibitors and obtained the expected pharmacology. A key advantage of this approach is that it does not require cell engineering. Therefore, the phosphorylation or total level of an endogenous protein can be detected in any cell type. Moreover, Lumit technology was configured in an assay that monitors the SARS-CoV-2 Spike RBD and human ACE2 protein-protein interaction (PPI) to screen for inhibitory molecules of virus entry as well as the characterization of clinical specimens that may contain virus neutralizing antibodies (Nabs). The assay utility for the discovery of novel inhibitors was demonstrated with a panel of anti-RBD antibodies, ACE2-derived miniproteins and soluble ACE2. Studying the effect of variants RBD mutations on ACE2 binding using this assay showed that the RBD N501Y mutation increased RBD affinity toward hACE2 tenfold. Also, neutralizing antibodies (Nabs) from COVID-19 positive samples were successfully detected and the results further suggest the persistence of Nabs for at least 6 months. This method could have broad utility, from streamlining the analysis of signaling pathways of interest, identification of target-specific chemical degraders or biologic inhibitors to enabling the screening of novel PPI inhibitors.